Most current conventional methods of the proteome analysis focus on measuring and recording variations in protein level. These approaches are commonly referred to as “proteomics.” In general, proteomics seeks to measure the abundance of broad profiles of proteins being present in complex biological mixtures. A major goal of proteomics is to develop global methods for the analysis of protein function in samples of high biological complexity.
In typical proteomic experiments, the expression levels of proteins in cells, tissues, and/or fluids are compared using techniques such as two-dimensional electrophoresis or isotope-coded affinity tagging, where variations in protein abundance are used to infer changes in protein activity. However, many proteins are regulated by a complex array of post-translational mechanisms. For such proteins, alterations in their abundance may not correlate with changes in activity. For example, some proteins, such as metalloproteases (MPs) enzymes, are subject to numerous forms of post-translational regulation in vivo, including production as inactive zymogens and inhibition by endogenous proteins. These post-translational events hinder the functional analysis of MPs using conventional, abundance-based genomic and proteomic methods.
To address the problems that may be caused by the post-translational events, a chemical methodology known as activity-based protein profiling (ABPP) has been introduced. According to the ABPP method, active site-directed probes are used to record variations in the activity of proteins in whole proteomes.
ABPP probes typically include three moieties: a binding group that promotes interactions with the active sites of specific classes of enzymes, a reactive group that covalently labels these active sites, and a reporter group (e.g., fluorophore, biotin) for the visualization and affinity purification of probe-labeled enzymes. ABPP probes are used to tag specific groups of proteins based on functional properties rather than expression level alone, thus providing good access to low abundance proteins in complex proteomes.
The need to devise methods of measuring protein activity, as opposed to abundance are best illustrated in case of enzymes which are an important subset of proteins. Enzymes are known to be key to almost every biologic process, including blood coagulation, inflammation, angiogenesis, neural plasticity, peptide hormone processing and T-lymphocyte-mediated cytotoxicity. Several human diseases are known to be associated with dysfunctions in enzymes. These include, but are not limited to, hemorrhagic disorders, emphysema, arthritis and even to cancer.
To date, ABPP probes have been developed for many biomedically relevant enzyme classes, including serine hydrolases, cysteine proteases, and oxidoreductases, as well as for profiling enzyme activities in living cells and animals. Despite these and other advantages of ABPP over conventional proteomic methods, several important enzyme classes remain unaddressed by this approach. One class of enzymes for which the ABPP method has not been developed includes the metalloproteases (MP) comprising a large and diverse group of enzymes that play key roles in many physiological and pathological processes, such as tissue remodeling, peptide hormone signaling, and cancer.
One approach for the activity-based profiling of proteins that has been used is the creation of ABPP probes designed to target conserved nucleophiles in the protein active sites. For example, this method has been used for some classes of proteases, such as serine or cysteine proteases. However, this technique cannot be directly applied to MPs, which utilize a zinc-activated water molecule (rather than a protein-bound nucleophile) for catalysis. Therefore, an alternative approach is required to generate chemical probes that label the active sites of MPs with sufficient potency and specificity to enable functional profiling of these enzymes in whole proteomes.
In view of the foregoing, an acute need exists to develop activity-based protein profiling methods for metalloproteases. Embodiments of the present invention provide such methods.